The mean reference GFR was 60.5 ± 33.4 mL/min/1.73 m2. The bias (mean error) values for the estimated GFR from the CKD-EPI equation, BSA-unadjusted CKD-EPI equation, re-expressed MDRD study equation with the Thai racial factor, and Thai eGFR, 2012 CKD-EPI creatinine-cystatin-C, Cockcroft-Gault, and Janowitz and Williams equations were −2.68, 1.06, −7.70, −8.73, 13.37, 1.43, and 2.03 mL/min, respectively, the precision (standard deviation of bias) values were 6.89, 6.07, 14.02, 11.54, 20.85, 10.58, and 8.74 mL/min, respectively, and the accuracy (root-mean square error) values were 7.38, 6.15, 15.97, 14.16, 24.74, 10.66, and 8.96 mL/min, respectively. In conclusion, the estimated GFR from the BSA-unadjusted CKD-EPI equation demonstrated the least bias along with the highest precision and accuracy. Further studies on the outcomes of anti-cancer drug dose adjustments using this equation versus the current standard equation will be valuable.The accuracy of the estimated glomerular filtration rate (eGFR) in cancer patients is very important for dose adjustments of anti-malignancy drugs to reduce toxicities and enhance therapeutic outcomes. Therefore, the performance of eGFR equations, including their bias, precision, and accuracy, was explored in patients with varying stages of chronic kidney disease (CKD) who needed anti-cancer drugs. The reference glomerular filtration rate (GFR) was assessed by the  A precise GFR assessment is fundamental to several aspects of cancer therapy, including chemotherapy dose adjustment, decisions regarding surgery eligibility with perioperative management, and preparation of long-term care. An underestimated GFR in a patient with cancer could lead to inappropriate care, such as in the case of a patient being deemed ineligible for both medical chemotherapy and surgical treatment because their GFR is too low. Conversely, overestimation of the GFR could put a patient at unnecessary risk of drug overdose and unfavorable complications. Because most cancer chemotherapeutic agents are excreted mainly through the kidneys, the accuracy of the estimated glomerular filtration rate (eGFR) in patients with cancer is crucial to balancing treatment efficacy and the risk of adverse events. Although the eGFR calculated from serum creatinine (SCr) is widely used in general practice, overestimation of the GFR due to a patient’s reduced muscle mass and food intake due to malignancy is very common. Despite the increased accuracy of eGFRs obtained by determining the measured GFR (mGFR) using the clearance of exogenous filtration markers, this method has not been widely used due to the necessity of a 24-hour urine collection. Indeed, a standard reference for the eGFR for chemotherapeutic agent dose-adjustments remains undecided. The International Society of Geriatric Oncology preferred eGFRs using the Modification of Diet in Renal Disease (MDRD) equation over the Cockcroft-Gault equation for patients over 65 years old. Nevertheless, the equation from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) in a recent large-scale, retrospective study appeared to be superior to the MDRD equation for the eGFR assessment in patients with cancer3.The coexistence of chronic kidney disease (CKD) and cancer is common due to the increased incidence of cancer in patients with CKD4. Similarly, the Society of Geriatric Oncology guidelines prefer mGFR in anti-cancers that are mainly excreted through the kidney (or with apparent nephrotoxicity) or in cases of possibly inaccurate eGFRs5. It is important to recognize that both the MDRD and the CKD-EPI equations were developed using data from CKD patients, from which patients with malignancy are excluded11. Patients with malignancy, in contrast to those with CKD alone, mostly suffer from more severe sarcopenia, resulting in lower SCr (due to less creatinine production) and overestimated GFRs. As such, accurately estimated renal function is likely necessary for the proper adjustment of cancer chemotherapy. Although the MDRD and CKD-EPI equations are widely used to predict the GFR in the United States and Europe, some corrections are necessary for other ethnic groups, as is the case for the coefficient factor for the isotope-dilution mass-spectrometry (IDMS) tracer in the re-expressed MDRD equation proposed as 1.129 for the Thai population10. However, the validation of the eGFR derived from these equations in Thai patients with cancer has not yet been investigated.In general, a consensus of the international guideline group of KDIGO  recommends creatinine-based equations for initial testing, with other confirmatory tests for the estimation of kidney function in CKD patients14. In addition, increased serum CysC levels found in both solid and hematologic malignancies are likely related to the tumor’s nature as a cysteine protease inhibitor17. No studies have examined whether an equation based on serum CysC would improve GFR estimation in cancer patients compared to the estimates obtained by other equations. Therefore, the aim of the present study was to investigate the agreement and precision of the currently published eGFR formulae, including the CKD-EPI7, the body surface area (BSA)-unadjusted CKD-EPI, the re-expressed MDRD study equation with the Thai racial factor18, the Thai eGFR10, the 2012 CKD-EPI creatinine-cystatin C13, the Cockcroft-Gault19, and the most recent cancer patient-derived eGFR equation by Janowitz and Williams3, compared to the standard GFR measurement by 99mTc-DTPA.Moreover, serum cystatin C (CysC) is considered a potential replacement for SCr as a filtration marker, but the correlation between the eGFRs derived from CysC and SCr are still uncertain4. The exclusion criteria were as follows: i) history of active medical or surgical treatment for related malignancy within the past 6 months; (ii) acute deterioration of malignancy or related complications, including gastrointestinal bleeding, infection, severe malnutrition with an edematous state, acute kidney injury superimposed on CKD, congestive heart failure, and arterial or venous thrombosis; (iii) dialysis dependence; (iv) amputation; (v) breastfeeding or pregnancy; (vi) end-of-life status; (vii) current hospitalization; and (viii) current use of medications with SCr interference, including ascorbic acid, corticosteroids, trimethoprim, cimetidine, flucytosine, methyldopa, and levodopa.The study was performed in compliance with the Helsinki Declaration. All participants were informed and provided written informed consent to participate in this study, which was approved by the Human Research Ethics Committee of Chulabhorn Research Institute (No. 017/2559) and local institutional review boards. The inclusion criteria were adults aged 18–70 years old with the following conditions: (i) pathologically or cytologically proven solid or hematologic malignancy with a performance status according to the Eastern Cooperative Oncology Group (ECOG) of 0–1 and ii) CKD in stable condition at various stages (G1–G5) according to the KDIGO criteria99mTc-diethylene triamine penta-acetic Acid (99mTc-DTPA) plasma clearance method with a radiopurity of >95% and the percentage bound to plasma protein <5%. The reference GFR by 99mTc-DTPA plasma clearance was read by a radiologist who was blinded to the clinical data. All participants were measured for plasma radioactivity of 99mTc-DTPA at 5, 30, 60, 120, 180, and 240 minutes after a single intravenous bolus of 99mTc-DTPA, following the institutional protocol. Then, plasma radioactive activities were plotted as a function of time to create a time–activity curve to calculate the GFR normalized by BSA20 as well as the measured GFR (mGFR) according to the following equation :21The reference GFR in the present study was determined by the ® 400 plus autoanalyzer  adjusted with a traceable high-level IDMS reference. Serum cystatin C (CysC) was measured by an automated particle-enhanced turbidimetric immunoassay (PETIA) on an ARCHITECT AEROSET analyzer . The coefficient of variation for the serum CysC assay was 2.1%. Both SCr and serum CysC were measured within 30 days of the 99mTc-DTPA–reference GFR measurement. No patient-identifiable data were used. Anonymized data included age, sex, height, weight, BSA, blood pressure, SCr, serum CysC, and serum albumin, all measured on the same day. Body composition was assessed by bioimpedance analysis using a Body Composition Analyzer .The serum creatinine (SCr) of individuals was evaluated by an enzymatic assay with the COBAS INTRGRA2” without the necessity for BSA adjustment in calculated eGFR values”. The units of the Janowitz and Williams equations and estimated creatinine clearance by Cockcroft-Gault equation express as “mL/min”19. In addition, a BSA-unadjusted GFR for those equations are calculated by the following formulae: BSA-unadjusted GFR (mL/min) = eGFR (mL/min/1.73 m2) x [BSA (m2)/1.73].Seven different commonly used methods of GFR estimation were tested in this study, including the re-expressed MDRD study equation with the Thai racial factor, the CKD-EPI equation with and without the BSA adjustment, and the 2012 CKD-EPI creatinine-cystatin C, Cockcroft-Gault, Thai eGFR, and Janowitz and Williams equations, as shown in Table t-test or the Mann–Whitney U test and the χ2 or Fischer’s exact test were conducted to compare continuous variables and categorical variables, respectively. Bland-Altman plots were used to assess the agreement between the reference GFR and eGFR22. The difference between the reference GFR and eGFR (reference GFR minus eGFR) was also calculated. The performances of the eGFR equations were evaluated for bias and precision. Bias measurements were expressed as the mean error (ME)23. Meanwhile, precision was defined as the standard deviation (SD) of the mean absolute difference24. Accuracy was defined as the root-mean square error (RMSE), which was calculated according to the following formula (n represents the sample size):25The baseline characteristics of the patients are presented as the mean ± standard deviation (SD). Other data are presented as median ± interquartile ranges (IQR). Student’s p-value < 0.05 was considered a statistically significant difference.In addition to the RMSE, the accuracy of the equations was also calculated using the percentage of the eGFR falling within the range of 10%, 15%, and 30% of the reference GFR. Statistical analyses were performed using STATA version 13.1 . A 99mTc-DTPA clearance (the reference GFR) was 50.4 mL/min/1.73 m2 (interquartile range [IQR] from 32.6 to 86.6 mL/min/1.73 m2), with almost 80% of patients categorized with stages G1–G3b of chronic kidney disease (CKD) according to the KDIGO classification. Notably, there was no participant with an extreme GFR  during the observation period. The average body mass index (BMI) and body surface area (BSA) were 21.6 ± 3.1 kg/m2 and 1.68 ± 0.2 m2, respectively. The mean serum creatinine (SCr) was 2.5 ± 1.6 mg/dL . In addition, the median muscle mass and soft lean mass were 22.5 ± 5.8 and 39.6 ± 8.7 kg, respectively, which was significantly lower than those in normal-weight and lean populations26 (p < 0.01).The patient characteristics are summarized in Table (Table The performances of seven published models of estimated GFR (eGFR) (see Methods) in patients with cancer were compared with those of the reference GFR. Among them, the eGFR from the BSA-unadjusted CKD-EPI equation demonstrated the greatest accuracy according to the root-mean square error (RMSE)  with the least bias . The novel model of the Janowitz and Williams equation was the third most accurate and least biased model for the eGFR; the RMSE and ME were 8.96 mL/min  and 2.03 mL/min , respectively. For the re-expressed MDRD study equation with the Thai racial factor, the RMSE and ME were 15.97 mL/min  and −7.70 mL/min , respectively. For the Thai eGFR equation, the RMSE and ME were 14.16 mL/min  and −8.73 mL/min , respectively. Notably, the 2012 CKD-EPI creatinine-cystatin C equation demonstrated the most bias with the least accuracy compared with the other equations . Regarding sex differences, the BSA-unadjusted CKD-EPI equation demonstrated the most homogeneity between male and female patients compared to the other eGFR equations. Notably, the Thai eGFR equation clearly demonstrated an overestimation of GFR in males in comparison to that in females .The agreement between the measurements by Bland-Altman and residual plots indicated that the BSA-unadjusted CKD-EPI equation showed the most accurate, least biased, and least heteroscedastic results, i.e., the most constant variance in different subpopulations, compared to those from the other equations Fig. . Regardiles Fig. .Figure 1(Table ), it underestimated the GFR, particularly in patients with GFR ≥60 mL/min . Similar to the BSA-unadjusted CKD-EPI equation, both the Janowitz and Williams equation and the CKD-EPI equation yielded estimates that were compatible with the reference GFR  in all GFR ranges. Meanwhile, both the Thai eGFR equation and the re-expressed MDRD study equation with the Thai racial factor demonstrated overestimation of the GFR in advanced CKD .We also investigated the utility of these eGFR equations with the reference GFR in the CKD population grouped according to GFR range as following: i) GFR ≥60 mL/min, ii) GFR 30–59 mL/min, and iii) GFR <30 mL/min. As shown in Fig. Table , it undemin Fig. . SimilarCKD Fig. .Figure 2The performances of the published models were also analyzed according to the KDIGO classification (CKD stage G1–G5), as shown in Table Our study showed that the body surface area (BSA)-unadjusted CKD-EPI equation showed the best performance for GFR estimation in terms of both precision and accuracy, followed (in order) by the CKD-EPI equation as well as the Janowitz and Williams equation for patients with cancer, the Cockcroft-Gault equation, and the Thai eGFR equations. Meanwhile, the 2012 CKD-EPI creatinine-cystatin C equation was the least precise and the least accurate eGFR equation in cancer patients as determined by the standard deviation of the absolute difference and root-mean square error (RMSE), respectively.27, and patients with cancer commonly present with impaired renal function28. At present, there are three most commonly used formulae in oncology worldwide—the Cockcroft-Gault, the MDRD study, and the CKD-EPI equations19—as well as the Thai eGFR equation, which is being adopted in practice nationwide10. While the CKD-EPI equation is recommended for use in routine clinical practice by the KDOQI and the National Kidney Foundation (NKF), most cancer centers use the MDRD study equation, following the International Society of Geriatric Oncology recommendation5. Nevertheless, the CKD-EPI equation is more accurate than the MDRD study equation in patients with reduced muscle mass, as eGFRs of 45–60 mL/min/1.73 m2 estimated by the MDRD study equation might be estimated as above 60 mL/min/1.73 m2 by the CKD-EPI equation11. Moreover, Asians have been shown to have a higher percentage of body fat for the same level of BMI than Caucasians, suggesting lower levels of muscle mass;29 this suggests ethnic interference and the necessity for robust validation of eGFRs in patients with cancer.GFR is currently the standard measurement for determining renal function; it was less biased than the Cockcroft-Gault equation and the re-expressed MDRD study equation with the Thai racial factor. Although the Cockcroft-Gault equation demonstrated the second least bias of the eGFR equations (mean error 1.43 mL/min), the precision and accuracy were less than the those of BSA-unadjusted CKD-EPI, CKD-EPI, and Janowitz and Williams equations (Table 3). The re-expressed MDRD study equation with the Thai racial factor, a preferable equation for CKD in the Thai population10, showed widest bias in eGFRs < 60 mL/min with a tendency of overestimation , possibly due to sarcopenia in patients with cancer30. Indeed, the participants in the present study had an 8.5% lower mean muscle mass compared to those of patients with HIV infection , another chronic illness population31. Additionally, the BSA-unadjusted CKD-EPI equation would be more applicable than the re-expressed MDRD study equation for calculation of eGFR in cancer patients with higher sensitivity and specificity in CKD determination, particularly in patients with CKD stage G2–G5 (Table 5). However, the use of BSA in corporation with eGFR formulas should be interpreted with caution particularly in CKD stages of KDIGO because the unit of eGFR in KDIGO naturally presents as mL/min/m2 4. In other words, there must be no difference between BSA-adjusted equations and BSA-unadjusted equations in term of the KDIGO guideline.In our study, the BSA-unadjusted CKD-EPI equation was the least biased equation Figs.  and 2; i32, the 2012 CKD-EPI creatinine-cystatin C eGFRs had low precision and accuracy in our results, possibly due to the lack of patients with cancer during the standardization of this equation33. Interestingly, the Thai eGFR equation demonstrated better performance than the 2012 CKD-EPI creatinine-cystatin C equation and the re-expressed MDRD study equation with the Thai racial factor, possibly due to the increased generalizability to the CKD population of the Thai eGFR equation and/or the different methods used for the reference GFR determination10. A further validation study in patients with cancer might be necessary to identify proper serum CysC-based and/or SCr-based eGFR equations for the Thai population. Moreover, the spread of bias among the BSA-unadjusted CKD-EPI, CKD-EPI, and Janowitz and Williams equations from the reference GFR was evenly distributed  despite the increased deviation from the reference in patients with eGFR <60 mL/min with the Janowitz and Williams equation . This phenomenon might be explained by the low sensitivity for advanced CKD stage with the Janowitz and Williams equation. Although the Janowitz and Williams equation is a somewhat sophisticated mathematical formula and is weak in its assessment of advanced CKD, it was impressive in assessments of early-stage CKD and is available as an online calculation tool (http://tavarelab.cruk.cam.ac.uk/JanowitzWilliamsGFR/)34.Although the 2012 CKD-EPI creatinine-cystatin C equation was favorable in conditions of low SCr production, such as in the case of loss of muscle mass from limb amputations or neurological diseasesion Fig. . This ph35. Although there is currently no guideline consensus which method of eGFR is preferred in cancer patients, our findings are consistent with the most recent study by Janowitz and colleagues3, which demonstrate better predictive performance of the BSA-unadjusted CKD-EPI over the CKD-EPI equation. While the CKD-EPI equation is recommended for use in routine clinical practice by the KDOQI and the National Kidney Foundation (NKF), the CKD-EPI equation showed less accuracy compared with the BSA-unadjusted CKD-EPI in the present study. This paradox could be explained by the fact that the CKD-EPI equations included populations with mean BSA of 1.93 ± 0.2 m2 and BMI of 28 ± 6 kg/m2, reflecting the large number of overweight participants in the CKD-EPI study7. Interestingly, Levey et al.7 also reported the mean measured GFR of their studied CKD patients of 68 mL/min/1.73 m2 and the mean BSA-unadjusted measured GFR of 75.9 mL/min. Accordingly, the difference of 7.9 mL/min was found after reversing the BSA indexing process among their studied population. In the present study, the CKD-EPI equation showed greater performance over the Janowitz and Williams equation, particularly in CKD with GFR < 30 mL/min, possibly due to (i) the ethnic difference10, (ii) the higher proportion of patients with low muscle mass (and BMI) in our study, iii) the difference in reference eGFR (99mTc-DTPA plasma clearance in the present study versus three different time points of chromium-51 EDTA (51Cr-EDTA) administration in the other study) and iv) the inclusion criteria including both solid and hematologic malignancy in the present study3.Given the validation of several common eGFR calculations, the CKD-EPI equation (regardless of BSA adjustment) is the most appropriate for determining the CKD stage in patients with malignancy . Our findings support the 2016 cancer chemotherapy guidelines for treatment of renal injury, which states that i) eGFR (or creatinine clearance) without correcting BSA is used for drugs that the doses are fixed (BSA independent) and ii) eGFR (or creatinine clearance) corrected for BSA is used for drugs that the dose depends on BSA99mTc-DTPA method may overestimate GFR, particularly in patients with lower BMI36, the comparable inulin method for CKD patients has been mentioned in a large study37. Second, the performance status of most participants was good (ECOG 0–1) due to ethical restrictions. Patients with cachexia might have displayed more deviations in GFR. Third, a small number of patients with paraproteinemia—a disease with low SCr—were included in the present study. However, the exclusion criteria in this study ruled out most of the potential cofounding factors influencing the eGFR assessment. Fourth, the impacts of the different eGFR equations on clinical outcomes, complications, and other aspects of renal dysfunction  and comparisons of the use of eGFR with the use of actual (reference) GFR were not explored. Further studies are warranted.There were several limitations in our study. First, the gold standard renal inulin clearance was not included in the present study. Although the Taken together, we propose that the BSA-unadjusted CKD-EPI formula is the most favorable eGFR equation in patients with cancer, followed by the CKD-EPI and the Janowitz and Williams equations. Further validation studies with pharmacokinetic exploration are of interest.


Several typhoons around the China Seas were captured by Chinese GF-3 SAR in 2017; e.g., Noru, Doksuri, Talim and Hato. The wave fields simulated from the third-generation numerical wave model WAVEWATCH-III (WW3) are collocated with these images. In general, the distribution patterns of the typhoon waves from the WW3 model are consistent with wave fields from the European Centre for Medium-Range Weather Forecasts (ECMWF) at 0.125° grids, indicating that the simulation results from the WW3 model are suitable for our study. In addition to winds retrieved from GF-3 SAR images in vertical-horizontal (VH) polarization, the characteristics of the typhoon wave on vertical-vertical (VV) polarization GF-3 SAR images are studied. It is found that SWH has a linear relationship with NRCS and cvar, however, SWH fluctuates with wind speed at all incidence angles. Based on the analyzed results, we simply tune two empirical wave retrieval algorithms for GF-3 SAR in typhoons. Although the correlation (COR) reaches 0.5 taking account into the NRCS term, a more accurate retrieval algorithm, including more related terms, is anticipated for further development for GF-3 SAR and validated through more typhoon images.The motivation of this work is to explore the possibility of typhoon wave retrieval (the main parameter is significant wave height (SWH)) for C-band Gaofen (GF-3) synthetic aperture radar (SAR) with a wide swath coverage (>400 km). We aim to establish an analysis of a typhoon wave in the subresolution-scale (approximately 20 × 20 km Typhoons have a significant influence on the heat, water vapor, and material exchange between the ocean and the atmosphere, and they are considered to be an important dynamic phenomenon of oceanographic study. Due to the risks involved in measuring waves during a typhoon, it is rather difficult to use traditional technology; e.g., moored buoy and sailing ship. Space-borne synthetic aperture radar (SAR) can obtain high spatial resolution information of the sea surface with a large swath coverage following its orbit. Therefore, currently SAR is an important, direct, and effective technical way to obtain marine data . MoreoveTo date, theoretically-based wave retrieval algorithms have included the “Max-Planck Institute” algorithm (MPI) ,6,7, theWind is an essential parameter in typhoons. The geophysical model function (GMF) is widely used for wind retrieval from SAR. GMF at C-band includes CMOD4 , CMOD-IFThe Chinese Gaofen-3 satellite carrying a SAR sensor at C-band was launched by the China Academy of Space Technology (CAST) in August 2016. During a mission in 2017, data from several typhoons were captured and recorded in the China Sea; e.g., Noru, Doksuri, Talim and Hato. These images were acquired in dual-polarization (VV and VH) and officially delivered by the National Satellite Ocean Application Service (NSOAS). It was found that the SAR backscattering signal encountered a saturation problem under strong wind conditions (probably greater than 25 m/s) , while tIn our work, five VV-polarization GF-3 SAR images including apparent typhoon eyes (TE) have been compared with computations from the numerical wave model WAVEWATCH-III (WW3). Through the collocated dataset, the relationships between significant wave height (SWH) and several SAR-measured parameters were investigated, including strong winds retrieved from VH-polarization GF-3 SAR images, variance of the normalized SAR image , and normalized radar cross section (NRCS). These are related with the sea state and can be obtained without visible wave streaks on the SAR images. As a preliminary study, we propose two empirical wave retrieval algorithms for GF-3 SAR in typhoons.The remaining parts of this paper are organized as follows: Data collection are described in DN is SAR-measured intensity, M is the external calibration factor and N is the offset constant stored in the annotation file. It should be noted that those images acquired in GLO and WSC mode comprise several strip-beams that are slightly lighter near their edges because of the effect of instrumental noise. Although there are a few time differences between the SAR imaging time and the track data, it is shown that GF-3 SAR has the ability to capture typhoons. In this study, five GF-3 SAR images acquired in Global Observation (GLO) and Wide ScanSAR (WSC) mode are used. These images were taken in dual-polarization (VV and VH) and processed to a Level-1B (L-1B) product during the period of typhoons Noru, Doksuri, Talim, and Hato in 2017, when the maximum wind speed reached 40 m/s. The quick-look images of the collected images in VV-polarization channel after calibration are presented in We also show the five GF-3 SAR images in the VH-polarization channel corresponding to each image in In order to study the performance of typhoon waves on GF-3 SAR, we initially collected the wave data from European Centre for Medium-Range Weather Forecasts (ECMWF) reanalysis datasets. Specifically, ECMWF continuously provide global atmospheric-marine reanalysis fields for the worldwide investigator, which have a fine spatial resolution (up to 0.125 × 0.125°) at an interval of 6-h daily. These data are considered to be reliable sources, which are widely used for developing and validating the algorithms for SAR retrieval; e.g., wind ,23 and wThe third-generation numerical wave model, abbreviated as the WW3 model, was developed by the National Oceanic and Atmospheric Administration/National Centers for Environmental Prediction (NOAA/NCEP), which is in the spirit of the previous WAM model. Recent research has revealed the WW3 model to have a good performance when simulating the characteristics of typhoon waves ,44. TherAs mentioned in the introduction, strong winds are an important contributor to typhoon waves. We briefly present the recent development for strong winds retrieval from VH-polarization GF-3 SAR images. In addition, typhoon waves at 0.1° grids, as simulated by the WW3 model, are shown.f2 is the function of radar incidence angle which is normalized into  and the coefficients α, β, A, B, and matrix C are the tuned constants.Through a collocated dataset, including three VH-polarization GF-3 images in GLO/WSC mode and wind simulations at 0.12° grids from GRAPES-TYM in typhoons, the dependence of wind speed and incidence angle on VH-polarization NRCS of GF-3 SAR was investigated, following which an empirical algorithm was developed in , which iThe proposed algorithm has been implemented for several additional VH-polarization GF-3 SAR images. The inverted winds were compared with measurements from Windsat winds at 0.25° grids and GRAPES-TYM winds with wind speeds up to 40 m/s. It is reported in  that theFive of those GF-3 SAR images were also taken in the VV-polarization channel with visible typhoon eyes (TEs). Therefore, the results proposed in  can be dIn this study, we employed the WW3 model to simulate wave fields in typhoons with fine resolution in spatial and temporal scale. The forcing fields are 0.12° gridded winds from GRAPES-TYM, which were used in our recent study for developing a strong winds retrieval algorithm . The daWe tried to collect the wave measurements from altimeter Jason-2 over the duration of the four typhoons; e.g., Noru, Doksuri, Talim, and Hato. Unfortunately, only the data of altimeter Jason-2 for typhoons Noru and Hato were available. As examples, the simulated SWH maps from the WW3 model taken on 5 August 2017 at 06:00 UTC and on 23 August 2017 at 17:00 UTC are shown in θ, which are assumed to be the basic variables in the empirical wave retrieval algorithms [The sea state SWHs are supposedly related with winds at 10 m above the sea surface, NRCS united in dB and dimensionless cvar at a fixed incidence angle gorithms ,15,16,172. Note that inhomogeneous sub-scenes due to heavy rainfall, where the ratio of image variance and squared image mean the value is greater than 1.05, are excluded [In order to obtain a reasonable dataset, the whole VV-polarization GF-3 SAR image is divided into a number of square sub-scenes, with a spatial coverage of about 20 × 20 kmexcluded ,42,46. TInterestingly, SWH has a linear relationship with NRCS and cvar, as shown in It appears that wave streaks are mostly invisible in a low spatial SAR image; e.g., more than 100 m for GF-3 SAR acquired in GLO and WSC mode—making it impossible to invert SAR intensity into an ocean wave spectrum under typhoon conditions. Considering the possible relationships mentioned above, a convenient approach for typhoon wave retrieval should be attainable, similar to the traditional GMF methodology for wind retrieval.a and b are the tuned constants, as shown in We simply propose two empirical wave retrieval algorithms for GF-3 SAR, taking a unqiue formulation as follows,A cutoff wavelength in the azimuth direction is equivalent to the effect of non-linear velocity bunching on SAR. Theoretically, a cutoff wavelength is positively related with sea state SWH ,6,17. Th2 spatial coverage extracted from those images are collocated with SWH from the WW3 model at a 0.1° grid. Through the dataset, we study the relationship between SWH and three SAR-derived parameters at incidence angles from 10° to 50° for a 10° bin; e.g., wind speed, NRCS and cvar. Interestingly, it is found that SWH is linearly related with NRCS and cvar, which agrees with the conclusions proposed in [Five GF-3 SAR images with apparent TEs during four typhoons are used in this study. The WW3 model is employed to simulate wave fields in those typhoons. The validation against measurements from altimeter Jason-2 shows a less than 0.5 m STD of SWH. A number of sub-scenes with a 20 × 20 kmposed in . TherefoIt is concluded that taking advantage of SAR-measured NRCS is a promising solution in the development of a typhoon wave retrieval algorithm. In fact, the case study in  for wave


The general characteristics of children with and without growth retardation were also compared. All participants received a full-calorie, traditional KD supplemented with vitamins, minerals, and citrate. Most children  treated with the KD did not show growth retardation at 12 months. Although participants with and without delay of growth did not differ in terms of baseline clinical characteristics, dietary prescriptions, or supplementation patterns, marked ketosis at 12 months tended to occur more frequently in the latter group. Altogether, our results indicate that growth retardation may occur in a minority of children treated with the KD. However, further research is required to identify children at risk and to clarify how increased ketones levels may affect endocrine pathways regulating growth during KD administration.Data on the impact of the ketogenic diet (KD) on children’s growth remain controversial. Here, we retrospectively investigated the occurrence of linear growth retardation in 34 children  diagnosed with drug-resistant epilepsy (DRE;  The ketogenic diet (KD) is not only considered a valuable non-pharmacological therapeutic option for children with drug-resistant epilepsy (DRE)  but it aKD requires constant nutritional monitoring over time both to ensure its effectiveness and to reduce the likelihood of short- and long-term adverse effects . Among tData on factors that may affect the growth of children treated with the KD  remain scarce. Peterson et al.  demonstrn = 14) or GLUT1-DS (n = 20) who had been treated with the KD for 12 months. The general characteristics of children with and without growth retardation were also compared. All of these studies have been conducted in patients with DRE. As far as GLUT1-DS is concerned, only one case of a child with a growth deficit related to growth hormone (GH) deficiency has been reported in the published literature . BecauseThe study protocol complied with the tenets of the Helsinki Declaration and was approved by the ethical committee of the Fondazione IRCCS Policlinico San Matteo of Pavia on 12 November 2018 (code number: 20180083746). All patients and/or caregivers provided their written informed consent.Inclusion criteria were as follows: 1) age between 2 and 17 years; 2) diagnosis of DRE (defined as unresponsiveness to at least two anticonvulsant medications) or GLUT1-DS; and 3) treatment with KD for at least 12 months. All participants were consecutively enrolled at the Department of Child Neuropsychiatry of the IRCCS Casimiro Mondino Foundation  between 2008 and 2017. Children with severe organ failure, thyroid disorders, or requiring enteral and parenteral nutrition were excluded. The ambulation status of each child was analyzed in relation to age and physical activity. Data were extracted from a retrospective review of clinical records and entered onto an electronic database. Clinical, anthropometric, growth, and body composition parameters  of the study participants were collected at baseline and at a 12-month follow-up. KD prescriptions and blood β-OHB concentrations were also recorded. Using a standardized approach , the HumOral citrates were also administered to prevent the formation of kidney stones. All patients were started at home on a 1:1 ketogenic diet, with ketogenic ratios subsequently increased to 2:1, 3:1, or 4:1 in order to achieve blood β-OHB levels ≥2.0 mmol/L. Family members were instructed to monitor blood β-OHB concentrations twice per day during the induction phase and report values by e-mail to the study investigators. At follow-up visits, each patient underwent 1) measurements of fasting blood ketones, 2) assessments of compliance to the prescribed diet regimen and supplement use, and 3) screening for potential adverse effects. Anthropometric measurements taken by trained dieticians were used to define growth patterns in accordance with standard procedures . Body weHeight velocity (cm/years) was estimated from height measurements obtained immediately before starting the diet and 12 months thereafter. Reference values for age and sex were obtained from Tanner et al. . In keepp values <0.05 were considered statistically significant. Continuous variables were expressed as medians and interquartile ranges (IQRs) and compared across the three growth groups using the non-parametric Kruskall-Wallis test followed by post hoc non-parametric sign tests with the Bonferroni’s correction. Categorical variables were analyzed with the chi-squared test or the Fisher’s exact test, as appropriate. Associations between changes in height z-scores at 12 months and other continuous variables were tested with the Spearman’s correlation coefficient (ρ). All calculations were performed with STATA/SE® for Windows, version 15 . Two-tailed p = 0.041) and began dietary treatment earlier after diagnosis —an expected finding being KD the only available treatment for GLUT1-DS.Patients with GLUT1-DS were diagnosed at an older age  than those with DRE . There were significant differences in terms of anti-epileptic drugs (AEDs) and ambulation between the two groups. Specifically, all patients with GLUT1-DS were able to ambulate and none of them was taking AEDs. Ten patients with DRE were on multiple anti-epileptic drugs (AEDs) and their pharmacological treatment was not modified throughout the study. Moreover, five of them were unable to ambulate.p = 0.675). No significant changes in all parameters emerged for both groups. Specifically, after 12 months of KD, the median height z-score did not change significantly from baseline both in patients with DRE  and GLUT1-DS . Based on these observations, all of the study patients were grouped together for analysis of growth patterns regardless of their underlying diagnosis. p = 0.028), which was lower in Group-D  than in Group-T .Group-I consisted of six children  of whom 3 had GLUT1-DS. Three children were aged between 2 and 6 years and 3 between 7 and 10 years. One child was unable to ambulate. Group-T included 21 patients  of whom 13 had GLUT1-DS. Eight children were aged between 2 and 6 years, 5 between 7 and 10 years, and 8 between 11 and 17 years. Three children were unable to ambulate. This group comprised three post-pubertal children. Finally, Group-D consisted of seven children  of whom 4 had GLUT1-DS. Five children were aged between 2 and 6 years and 2 between 7 and 10 years. One child was unable to ambulate. The baseline characteristics of the three growth groups are summarized in p = 0.043).A lower percentage of energy intake as carbohydrates was evident for Group-T  compared with Group-I . In the entire sample, a moderate inverse correlation was evident between fasting β-OHB blood levels at 12 months and the height z-score . Fasting β-OHB blood levels at 12 months did not differ significantly in the three study groups, being 1.8 mmol/L (IQR = 1.2–2.7 mmol/L) in Group-I, 2.4 mmol/L (IQR = 1.5–3.2 mmol/L) in Group-T, and 4.1 mmol/L (IQR = 1.9–6.1 mmol/L) in Group-D (p = 0.045) and growth velocity  z-scores were significantly lower in Group-D than in Group-I.p = 0.003) or Group-D .After 12 months of KD, weight z-score was found to be more markedly impaired in Groups-D and -T than in Group-I—as shown by the following significant intergroup differences in ∆ weight z-scores: Group-I  versus Group-T ] and did not appreciably change at 12 months. Fat mass percentage at baseline did not differ significantly in the three groups [Group-I , Group-T , and Group-D .In the entire sample at 12 months, there was a moderate positive correlation between height z-scores and weight z-scores  maintained or even improved their growth at 12-month follow-up. Notably, this observation was consistent in children diagnosed with both DRE (of whom 11 out of 14 maintained or improved growth) and GLUT1-DS (among whom 16 out of 20 maintained or improved growth). Our findings are in accordance with those obtained in recent long-term follow-up studies. Wibisono et al.  reportedTaken together, published evidence and our current data consistently indicate that growth retardation may occur in a minority of patients treated with the KD. From a clinical standpoint, it would be helpful to identify children at higher risk of poor growth before starting the KD. In an effort to address this issue, we compared the general characteristics of patients showing different growth patterns. However, Group-D was significantly different from other groups only in terms of age at diagnosis (which was found to be lower in this group compared to the Group-T). Because the interval between diagnosis and KD initiation did not show significant intergroup differences, the length of time during which the patient remained on a balanced diet cannot be a factor explaining the observed growth differences. Moreover, dietary prescriptions, supplementation patterns, and follow-up periods did not differ significantly in the three growth groups.Potential causes of poor growth in children on the KD include inadequate intakes of energy and protein , the effects of the underlying diseases and treatments, acidosis/ketosis and related endocrine changes.In a retrospective study conducted in 21 children (mean age: 7.9 ± 3.8 years), Williams et al.  reportedTo achieve a level of ketosis adequate for seizure control (in epilepsy) and for ketone use as an alternative fuel for brain metabolism (in GLUT1-DS), very low amounts of carbohydrates, adequate amounts of protein, and very high amounts of fat must be prescribed in a rigidly controlled ratio (ketogenic ratio)—resulting in a highly unbalanced diet compared to WHO recommendations for children and adolescents ,23,24,25The maintenance of the ketogenic ratio limits the amount of proteins that can be included in the classical KD—although an amount of 0.8–1 g/kg body weight is generally provided. However, the amount of proteins needs to be carefully evaluated according to energy requirements in terms of protein-to-energy ratio —an indexGrowth patterns in children on KD may be influenced by the underlying disease as well as the presence of comorbidities and concomitant drug treatments. Patients with DRE may show suboptimal growth patterns even before the initiation of KD because of endocrine dysfunction or the use of multiple AEDs ,33,34. HA single case of growth retardation related to GH deficiency has been reported in GLUT1-DS , making In the current study, growth deficit was identified in only 20% of the study participants, without significant differences between children with DRE and GLUT1-DS. With the exception of one child in Group-D who was non-ambulatory, the main characteristic of our patients with delayed growth was a reduction in weight z-score, suggesting that periods of inadequate caloric intake may have occurred notwithstanding an adequate calorie prescription based on actual needs. A high-fat KD may be unpalatable for some subjects and can result in occasional food refusal, which was more frequently observed in Group-D (3 out of 7 children). However, a reduction in z-score was also observed in the Group-T. More interestingly, a trend toward higher ketone levels was evident in Group-D, albeit not significantly so . A chronic systemic reduction of pH is known to impair growth  and KD mThe caveats of our investigation include its retrospective design and the limited sample size. These limitations notwithstanding, our study describes for the first time the growth patterns of children with GLUT1-DS who had been treated with the KD. Importantly, most of our patients were able to maintain or improve their growth patterns. We speculate that our promising results may be ascribed to an accurate assessment of energy needs , an adequate protein-to-energy ratio according to WHO recommendations, as well as to the continuing support offered to caregivers involved in the management of KD. Moreover, the routine supplementation of oral citrate may have mitigated the detrimental impact of chronic metabolic acidosis. However, more research is required to identify children at higher risk of growth delay. In addition, the potential role played by high ketone levels on altered IGF-1 signaling in influencing linear growth of children on KD needs further exploration. Under these circumstances, a more in-depth endocrine evaluation is advisable for all children who show evidence of growth retardation while on the KD.


Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain—L1-4, L66, L79—and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics. Therapeutic antibodies are the most important biopharmaceuticals, and their share in the market of all licensed drugs is continuously on the rise . In ordeThe biopharmaceutical properties of a therapeutic antibody can be optimized during formulation development by adjusting the buffer system and the pH, by including additives etc. However, stability and aggregation can be addressed during lead antibody optimization. This facilitates later development by enhancing the developability of the molecule. Stability and aggregation propensity have been improved by sequence- and structure-based approaches ,13,14. HThe Kabat numbering scheme was used for identification of antibody variable and constant domain residues . The antE. coli and purified as described [E. coli BL21  was transformed with the respective vector. Cells were lysed, and cell debris removed by centrifugation at 200,000× g for 20 min. Recombinant MIF was purified by application of the supernatent to DEAE-Sepharose FF  column followed by re-buffering in 20 mM Bis/Tris, pH 6.3 using a HiPrep 26/10 desalting column . A second purification step employed cation exchange chromatography using a Source 30S  column and elution of MIF with a linear salt gradient up to 100 mM NaCl.Recombinant MIF was expressed in escribed . In brie2SO4. The amount of chromogenic product formed was determined spectrophotometrically at 450 nm.MIF specific IgG titer was measured by enzyme-linked immunosorbent assay (ELISA). Typically, 96-well plates were coated with full length recombinant human MIF and incubated over night at +4 °C. Plates were then blocked by 1.5% fish gelatin diluted in phosphate-buffered saline (PBS), washed with PBS four times before applying samples in different dilutions. Samples were incubated 2 h at room temperature, the plates were washed four times with PBS, and human IgG was detected with a horseradish peroxidase-conjugated goat anti-human IgG . Finally, plates were washed four times with PBS before TMB  was added. After incubation in the dark at room temperature for 30 min the reaction was stopped by addition of 1.8 M H2SO4, and the reaction product was quantified as described above. Plasma concentration was calculated against standard curves of the test items. The human IgG ELISA was established as a fit for purpose assay.Human IgG concentrations were determined by a specific ELISA. Typically, 1 µg/mL of an affinity purified anti-human-IgG Fc goat F(ab’)2  was coated onto microtiter plates, blocked with 2% bovine serum albumine diluted in Tris-buffered saline with Tween 20 (TBST) for 1 h and washed four times with phosphate-buffered saline with Tween 20 (PBST) before applying samples in different dilutions. After 1 h incubation at room temperature, the plates were washed four times with PBST. Total human IgG was detected by a horseradish peroxidase–conjugated goat F(ab’)2, specific against the human F(ab’)2 . Subsequently, TMB was added and incubated at room temperature for 15 min. The reaction was stopped with 1.8 M HOxMIF and total MIF was quantitatively determined by a differential ELISA as described . EpitopeThe affinity of the antibodies was determined by surface plasmon resonance as described . In brieThe thermal stability of the antibodies was evaluated by differential scanning fluorimetry and differential scanning calorimetry. Differential scanning fluorimetry was conducted by using the Protein Thermal Shift assay (Thermo Fisher) according the instructions of the supplier. Fluorescence measurements were done on the 7500 Fast RT-PCR System (Thermo Fisher). The protein Thermal Shift Software (Thermo Fisher) was used for data analysis. The data analysis was based on the invariant calculation method for determining the midpoint temperature of the first unfolding transition. Differential scanning calorimetry was conducted using a NanoDSC-III  device with operation temperature range 25–100 °C and heating rate 2 °C/min in both the heating and cooling directions. After measuring both cells were filled with buffer and heated and cooled at the same settings as the sample run to obtain the buffer baseline. The buffer baseline was subtracted from the sample run and a polynomial baseline was generated to calculate the molar heat capacity.2PO4, pH 6.8, 150 mM Na2SO4, 0.02% NaN3, and 10% dimethyl sulfoxide. The column effluent was monitored continuously at 280 nm. The data were evaluated with a chromatographic data system . The performance of the whole system was checked and monitored by a system suitability test using a molecular weight standard .Time dependent aggregation/fragmentation of the antibodies were analyzed by SE-HPLC (Agilent) using a TSKgel G3000SW column . The column was eluted with 20 mM NaHn = 4 for each condition). Antibodies were administered intravenously at doses of 5, 10, or 30 mg/kg. The selected doses covered the range at which these antibodies inhibited the growth of xenograft tumors in nude mice [Animal experiments were carried out in accordance with the guidelines of the Medical University of Vienna  and were approved by the Animal Welfare Committee of the Medical University of Vienna and the Austrian Science Ministry. The pharmacokinetic characteristics of the anti-MIF antibodies were determined in male MF1 nude mice (ude mice . Blood wp < 0.05) in the fit. This was the case for all curves but those for 5 mg/kg parental BaxM159. Based on the bi-exponential decay model the following PK parameters were estimated: initial concentration, clearance (CL), area under the curve (AUC), terminal or beta phase half-life , and volume of distribution at steady state . The AUC was estimated from the time-concentration curves based on the trapezoidal rule from 0 to 144 h. CL was calculated from the relation CL = D/AUC, where D and AUC are administered dose and area under the curve, respectively. The VD,ss was calculated from the relation VD,ss = CL × MRT, where CL and MRT are clearance and mean residence time.Distributions were evaluated by the Kolmogorov-Smirnov test. If normal distribution was confirmed, data were evaluated by one-way ANOVA followed by Tukey’s post hoc test. Correlation analysis was based on Spearman’s correlation. Pharmacokinetic parameters were calculated by subjecting the data to a nonlinear least-squares fit to equations describing a mono- or a bi-exponential decay corresponding to a one- or two-compartment model, respectively. An F-test based on the extra sum of squares principle was used to confirm that the more complex model resulted in a statistically significant improvement (http://www.ncbi.nlm.nih.gov/igblast/)).It is hypothesized that germline V genes have been optimized during evolution for high expression and stability. Hence, a given antibody sequence should match the germline V genes as close as possible. Based on this approach we identified the germline VL and VH genes with the highest homology to the BaxM159 VL and VH sequence, respectively, by data base alignments  . Accordiab.org/; ). The moab.org/; . Moreoveis.org/; ). IndeedNext, we generated BaxM159 variants with DIQM1-4EIVL, A66G, or Q79E mutations, respectively, or combinations thereof . In totaD of all BaxM159 variants did not differ by more than a factor of two from that of the parental antibody BaxM159  as the parental antibodies [It is essential that the sequence-optimized BaxM159 variants recognize the target epitope with affinity and specificity similar to the parental BaxM159. Hence, we evaluated the BaxM159 variants for their binding to the target oxMIF by ELISA, their epitope specificity by monitoring binding to overlapping MIF-derived peptides and their affinity for oxMIF by surface plasma resonance. The equilibrium dissociation constant K BaxM159 . Similartibodies . Therefotibodies . Thus, aEIVLAQ), or at single position, i.e., A66G (4-DIQMGQ), and Q79E (5-DIQMAE). The variant 4-DIQMGQ with the A66G mutation was the only one of these three which increased the titer by factor 3–5 compared to the parental BaxM159 controls  increased the titer significantly by factor 4–10 compared to parental BaxM159. In contrast, the combination of mutants DIQM1-4EIVL and Q79E (7-EIVLAE) did not increased the titer significantly compared to parental BaxM159. Hence, the A66G mutation in the framework of BaxM159 seems to be crucial for increased expression of the antibody. The increase in expression was apparently caused by an increase in specific productivity (pg × cell/day) during the first five days of cell culture as evident from a 10 L batch run of 9-EIVLGE versus parental BaxM159 controls (Only BaxM159 variants which included the A66G mutation in combination with other mutations (9-controls .EIVLAQ) or A66G (4-DIQMGQ) increased the protein melting temperature Tm slightly compared to parental BaxM159 antibodies. However, mutation Q79E (5-DIQMAE) had by far the most Tm enhancing effect of the single region mutations and increased the Tm by >2 °C over the Tm of parental BaxM159.To assess the thermal stability of BaxM159 variants, antibodies were evaluated by differential scanning fluorimetry  and diffEIVLAE or 8-DIQMGE) did not increase Tm further compared to single mutation Q79E alone (5-DIQMAE). In contrast, combining DIQM1-4EIVL and A66G (6-EIVLGQ) or DIQM1-4EIVL, A66G and Q79E (9-EIVLGE) had a synergistic effect, i.e., increased Tm compared to the variants with mutations in a single region . Hence, 6-EIVLGQ and 9-EIVLGE had the highest Tm of all BaxM159 variants with an increase of >4 °C compared to the parental BaxM159. All findings, which were obtained by differential scanning fluorimetry, were confirmed by differential scanning calorimetry (Combinations of Q79E with DIQM1-4EIVL or A66G (7-orimetry .p = 0.043 based on Spearman’s rank-order correlation). This was also seen when the Tm-estimates obtained by differential scanning calorimetry were plotted against MIF-specific IgG titer (data not shown).It is reasonable to assume that the yield of functional protein is also determined by its stability. Accordingly, we examined if there was a correlation between MIF-specific IgG titer and the mean Tm of each BaxM159 variant: it is evident from the plot shown in EIVLGE for further characterization because of its desirable properties, i.e., it could be produced at high titers and it displayed high thermal stability. We monitored the propensity of variant 9-EIVLGE to form aggregates over a time period of 12 weeks at 4 °C in comparison to parental BaxM159: samples were analyzed by SE-HPLC after 0, 2, 4, 8, and 12 weeks  after intravenous injection of three different doses into MF-1 nude mice. Antibodies used in the pharmacokinetic study were polished by an additional cation exchange chromatography step resulting in removal of aggregates below 0.7% as confirmed by SEC-HPLC. Plasma samples were taken over a period of up to 144 h and analyzed for human IgG levels by ELISA. At equivalent doses, variant 9-EIVLGE reached about 10-fold higher initial concentrations in the blood than parental BaxM159 (2-DIQMAQ-K); this difference was maintained throughout the observation . In contrast, the terminal half-life t½, β of parental 2-DIQMAQ-K was only about 1.5 days. Accordingly, the clearance of parental 2-DIQMAQ-K was more than an order of magnitude larger than that of variant 9-EIVLGE. Conversely, the exposure (AUC) of variant 9-EIVLGE was about 20-fold higher than of parental 2-DIQMAQ-K  compared to parental 2-DIQMAQ-K. Similar differences were seen if the antibodies were administered by subcutaneous injection (data not sown). Furthermore, variant 9-EIVLGE exposure AUC was dose depended in the tested dose range whereas CL and VD,ss were dose-independent. In summary, variant 9-EIVLGE has superior pharmacokinetic characteristics compared to parental BaxM159 (2-DIQMAQ-K).We compared the pharmacokinetics of anti-MIF antibodies 9-fe t½, β  of aboutDIQMAQ-K . MoreoveEfficacy, safety, and developability of therapeutic antibodies depend—at least in part—on their biopharmaceutical properties such as stability, aggregation propensity, and expression levels. Here, we improved the biopharmaceutical properties of the anti-oxMIF antibody BaxM159 by introducing framework mutations.All BaxM159 variants with the individual mutations DIQM1-4EIVL, A66G, Q79E, or combinations thereof had the same oxMIF specificity, oxMIF affinity, and in vitro potency in a cell-based assay as the parental BaxM159. This is in line with a previous analysis of approximately 400 antibody structures . This stThe A66G mutation was the crucial determinant for an increase in titer, regardless of whether it was introduced as a single mutation or in combination with mutations in other regions. However, the mutation A66G improved stability only to a modest extent. A sequence analysis revealed that alanine at position 66 is rare (0.5%), whereas glycine is very common in both germline VL genes and rearranged antibody sequences. This might be due to a positive phi torsion angle at position 66 within this loop, which is feasible for glycine . The strEIVLAQ, 4-DIQMGQ, 5-DIQMAE), the Q79E mutation improved thermal stability most. The main-chain carbonyl group of E79 has been described to form a hydrogen bond with R61 of the LC chain [Of all single region mutations  resulted in further increase of thermal stability and expression. Indeed, the combination of all three mutation regions (9-EIVLGE) achieved the highest antibody titer and nearly the best stability. Similarly, combining selected framework (or CDR) mutations within the VH or VL domains has been described to improve biopharmaceutical properties additively or even synergistically [Combining the single region mutations  and the circulating levels of 9-EIVLGE was ≥10-fold increased. Possibly the higher propensity to aggregate and the lower stability of parental BaxM159 resulted in the enhanced formation of immune complexes, which are cleared rapidly. This interpretation is supported by the very large clearance and volume of distribution at steady state estimates for BaxM159. In contrast, clearance of 9-EIVLGE was ≥10-fold lower and the terminal half-live of 9-EIVLGE increased compared to parental BaxM159. In fact, 9-EIVLGE was cleared with elimination kinetics typical of IgG. It is likely that the improved pharmacokinetics of variant 9-EIVLGE are not due to different disposition of the two antibodies since healthy mice as used in this PK study do not express oxMIF and no target dependent interaction with murine leukocytes can be expected [EIVLGE might result in improved efficacy in vivo compared to the parental BaxM159 even if the in vitro potency of both molecules is similar.Based on the superior expression, stability, and aggregation propensity we focused on the BaxM159 variant 9-expected . Pharmacexpected , Fc engiexpected , but alsexpected . Here weFramework modifications have been shown to improve biophysical and biopharmaceutical properties of antibodies. Framework modifications can be based on knowledge-based rules, structural analysis, or statistical analysis of antibody sequences ,43,44,45–4, 66, and 79 of the VL domain did not amend oxMIF affinity, oxMIF specificity or function of the antibody. However, the modifications improved thermal stability, aggregation propensity, and expression. Moreover, the framework modifications resulted in reduced clearance, prolonged half-life and higher exposure. Germline-based framework modifications have therefore the potential to enhance both biopharmaceutical properties and pharmacokinetics of antibodies in a simple but efficient approach. Germline-based framework modifications were used to optimize the anti-oxMIF antibody BaxM159. The combined modifications at positions 1


Experimentally obtained non-coplanar 18-beam pinhole topographs were compared with computer simulations based on the Ewald–Laue theory. n-beam (n = 18) Ewald–Laue dynamical theory (E-L&FFT method). They were in good agreement with the experimentally obtained images captured using synchrotron X-rays. From this result and further consideration based on it, it has been clarified that the X-ray diffraction intensities when n X-ray waves are simultaneously strong in the crystal can be computed for any n by using the E-L&FFT method.Non-coplanar 18-beam X-ray pinhole topographs for a silicon crystal were computer simulated by fast Fourier transforming the X-ray rocking amplitudes that were obtained by solving the  This technique (E-L&FFT simulation) was reported by Kohn & Khikhlukha . Furthermore, the feasibility of calculating the X-ray intensities diffracted from a crystal that has plural facets, as shown in Fig. 9 of O et al. (2019), was discussed. In addition to this, if the E-L&FFT simulation could be performed even for a case where b) could be calculated. Here a large number (over 200) of reflected X-ray beams are simultaneously strong.The present authors have reported coplanar eight-beam pinhole topographs experimentally obtained and computer simulated by fast Fourier transforming (FFT) the rocking amplitudes calculated based on the ukha 2016 and Kohnukha 2016. In Okit al. 2019, it was 2.Fig. 2a). The exposure time was 300 s.In addition to the hexagonal six-beam topograph images, a further 12 images surrounding them were found on the IP as shown in Fig. 33.K  was calculated to be 1.7702394 Å−1 for a photon energy of 22.0 keV. The position of the Laue point La whose distance from reciprocal-lattice nodes 000, 440, 484, 088, K, was calculated on a computer. From Fig. 3a), other reciprocal-lattice nodes were likely to exist in the vicinity of the surface of the Ewald sphere; that is, their distance from La was approximately i.e.hkl to exist on the surface of the Ewald sphere. Here, a is the lattice constant of the silicon crystal, hkl from La were calculated in the range of i is the ordinal number of the reciprocal-lattice node in the first column of Table 1b). For obtaining this figure, a photon energy of 21.98415 keV was assumed. It was observed that the ith reciprocal-lattice nodes Laue’s fundamental equation of the dynamical theory  in O et al. 2019 was carried out with L = 50 mm and N = 4096.Furthermore, for the FFT to compute the E-L&FFT topographs, the description using equations (17)–(20) in O b) using one node (Intel Xeon E5-2680v3) of the supercomputer system ‘Sekirei’ of the Institute of Solid State Physics of the University of Tokyo. The calculation to solve the eigenvalue problem for a 36 × 36 matrix was several times as time-consuming as the coplanar eight-beam case solving the eigenvalue problem described with two 16 × 16 matrices described in O et al. 2019.It required 1080 s  to obtain the 18 topograph images shown in Fig. 34.c) shows the E-L&FFT simulated result with a photon energy of 21.98440 keV. In this figure, X-ray diffraction intensities due to the outer 12 reciprocal-lattice nodes on the blue circle in Fig. 4a). However, the outer 12 topograph patterns are almost unobservable when the energy deviation from a) was obtained.Fig. 6a) and 3b). There is remarkable consistency between the experimentally obtained and the E-L&FFT simulated images.Fig. 75.S(a)] (21.984150 keV). The vertical centre line in Fig. 8S(a)]. Further, an evident difference in the central part was observed between Fig. 7S(a)] and Fig. 8Fig. 8La and K , the situation where a large number of reciprocal-lattice nodes are simultaneously present in the vicinity of the surface of the Ewald sphere cannot be circumvented.In the present non-coplanar 18-beam case, the 18 reciprocal-lattice nodes are on two circles, drawn in red and blue in Fig. 4n has been removed completely from the n-beam E-L&FFT method to calculate the X-ray diffraction intensities. N is the number of reciprocal-lattice nodes present in the vicinity of the surface of the Ewald sphere whose presence should be considered. Another difficulty caused by the complex shape of the crystal has also been overcome with the description in O et al. 2019. Thus, the present authors could calculate the intensities of X-ray diffraction spots as shown in Fig. 1b) under the assumption that the crystal is perfect.However, the constraint on


G-protein coupled receptors (GPCRs) control multiple physiological states by transducing a multitude of extracellular stimuli into the cell via coupling to intra-cellular heterotrimeric G-proteins. Deciphering which G-proteins couple to each of the hundreds of GPCRs present in a typical eukaryotic organism is therefore critical to understand signalling. Here, we present PRECOG (precog.russelllab.org): a web-server for predicting GPCR coupling, which allows users to: (i) predict coupling probabilities for GPCRs to individual G-proteins instead of subfamilies; (ii) visually inspect the protein sequence and structural features that are responsible for a particular coupling; (iii) suggest mutations to rationally design artificial GPCRs with new coupling properties based on predetermined coupling features. They d G12/13 . Untangld G12/13 .Ligand binding to GPCRs induces conformational changes that lead to binding and activation of G-proteins situated on the inner cell membrane. Most of mammalian GPCRs couple with more than one G-protein giving each receptor a distinct coupling profile  and thusHere, we present a machine learning-based predictor (PRECOG) of Class A GPCR/G-protein couplings, which was developed as a part of the most systematic quantification of GPCR coupling selectivity to date . PRECOG GNA12 (Given one or more input sequences or Uniprot protein accessions (or gene symbols), PRECOG provides both overview predictions for each G-protein and putative mechanistic insights into how each prediction was made. Determinants of coupling-specificity are displayed on the sequence and on available (known or homologous) 3D structures. Users can also assess the impact of mutations on GPCR/G-protein coupling with respect to the wild type. We provide views that can aid users in selecting mutations that can help alter coupling specificity and ultimately to design receptors having specific couplings. We have already used PRECOG to predict coupling preferences of all human GPCRs, as well as to design a chemogenetic tool (DREADD) specific for GNA12 .GNAQ. Chimeric G-proteins are expressed in cells lacking endogenous Gα subunits  that mediate the AP-TGFα shedding response. This means that induction of specific GPCRs with titrated concentration of their ligands leads to binding to the co-transfected G-protein partner. AP-TGFα release signals over titrated concentrations were fitted with a sigmoidal concentration-response curve, from which we obtained EC50 and Emax values. For each chimeric Gα condition, an Emax/EC50 value was normalized by the maximum Emax/EC50 value among the 11 Gα chimeras . The baity, RAi . Functioity, RAi  through ity, RAi .q/11, Gi/o, Gs, G12/13) when speaking of the collective action of all proteins in each group, and gene symbols when referring to specific proteins. The 11 subunits grouped are: Gq/11 = GNAQ, GNA14, GNA15; Gs = GNAS, GNAL; G12/13 = GNA12, GNA13; Gi/o = GNAI1, GNAI3, GNAO1, GNAZ. We note that the six C-terminal sequences are identical for GNAQ and GNA11, and for GNAI1, GNAI2, GNAT1, GNAT2 and GNAT3 and that these members are not distinguished in our analyses.The use of individual genes instead of the standard coupling groups confuses nomenclature. For clarity, we use group symbols )  see Sup, using tP-value ≤ 0.05) among the coupled and uncoupled HMMs. Alignment positions with consensus columns  present in either HMMs, were considered as either insertion or deletion if they were present only in the coupled or not-coupled group. We also included length and amino acid composition of the third intracellular loop (ICL3) and C-terminus (C-term) considering features showing statistically significant differences  in coupled vs not-coupled.For a given G-protein, we then extracted positions showing statistically significant differences in terms of the amino acid bit-scores  scheme  to numbeADRB2-GNAS (PDB ID: 3SN6), ADORA2A-miniGNAS (6GDG), RHO-GNAI1 (6CMO), Oprm1-GNAI1 (6DDE), ADORA1-GNAI2 (6D9H), HTR1B-GNAO1 (6G79). For each complex, we aligned GPCR and chimeric Gα subunit sequences from the TGFα shedding assay to sequences homologous to the corresponding template structure chains. For each template structure, we calculated Z-scores and P-values (by generating 100 random permutations) for all the 144 Class A GPCR with each of the 11 G-proteins generating score distributions for coupled and uncoupled receptors to a particular G-protein and checking, through a Wilcoxon rank-sums test (P < 0.05), whether these were significantly different among the two groups. Whenever true, we considered that 3D complex as suitable to model the interaction with a particular G-protein and included derived Z-scores as features in the model.We integrated the above sequence-based feature set with additional structure-based features derived from available 3D complex structures of Class A GPCRs/G-proteins through the InterPreTS approach ,16, whicx1, x2, x3 …, xn are input features whereas w1, w2, w3, …, wn denote the regression coefficients. Thus, the probability of the input to couple with a given G-protein can be defined as:X and W denote the vector of input features  and of the regression coefficients , also termed weights, respectively.We implemented the predictor using a logistic regression (log-reg) classifier, available from the Scikit-learn package , consideRegularization is an essential technique in machine learning to counter over-fitting, which log-reg implements in two forms: L1 and L2. Both have a Lambda parameter that is directly proportional to the penalty of finding complex or over-fitted models. The regularization term (i) in the L1 form is the product of Lambda and the sum of the weights, while (ii) in the L2 form (used here) it is the product of Lambda and the sum of the squares of the weights. The target value is expected to be a linear combination of the features considered.c ∈ R∧n is the intercept, C is inverse of regularization strength (positive float), y takes values in {–1, 1} at trial i and n is the number of trials conducted. We used the liblinear method as the optimization algorithm as shown to be optimal for relatively small datasets  for the corresponding residue. For insertions or deletions, the approach returns the single bit score, derived from the respective HMM (i.e. coupled or not-coupled). If for any GPCR, no amino acid was present at the given position, it is assigned the highest bit scores from both the models, implying the least conserved scores.C (inverse of the regularization strength). Owing to the imbalance nature of the set, we set the class_weight parameter to balanced, which automatically adjusts the weights of the classes (coupled versus not coupled) inversely proportional to their frequencies in the training matrix. We divided the training matrix randomly into five equally stratified sub-matrices, preserving the ratio of positive (coupled) and negative (not coupled) GPCRs. To build a model for each G-protein, we chose the parameters showing the best Area Under the Curve (AUC) of the ROC curve. We repeated the experiments ten times, for each G-protein, to ensure minimal variance due to random division of the training matrix during cross validation. We assessed the performance of our predictor using standard metrics . From the coupled and not coupled HMMs of each of the 11 G-proteins, bit-scores of the corresponding amino acid at relevant positions are extracted and insertions/deletions are detected and used as features for predictions. Additional features are obtained by calculating the length and amino acid compositions (e.g. ICL3 and C-terminus).Given user input data, i.e. receptor WT or mutant sequences, the web server internally performs the following key steps to extract features  that predict the plausibility of interaction between the input and chimeric G-protein sequences according to available 3D complex structures (see above). For each 3D complex, InterPreTS takes the corresponding structure and multiple sequence alignments of receptors and G-proteins.Additionally, to detect the closest homolog for structural visualization purposes, every input sequence is aligned through BLAST  to 3D sthttp://flask.pocoo.org/), and the internal pipeline to handle back-end processes. Additionally, we used several JavaScript libraries at the front end. In more details, we used JSmol (http://www.jmol.org/) to view protein structure in 3D and neXtProt , mutations or FASTA sequences. A user can choose to make predictions or design a GPCR. The first option allows to predict the coupling preferences for input receptor(s), either wild type or mutant , i.e. the difference between Mutant and WT, and P(uncoupled), i.e. the difference between the WT and the Mutant. While the former tells if a particular mutation is predicted to increase a coupling of interest, while the latter suggests whether the same modification reduces unwanted couplings. To shortlist more interesting mutations, it is possible to set a threshold for both probability differences, which is by default 0.25, corresponding to the value that retains most of the interesting candidates based on our experience.The second option exploits feature information (i.e. weights) to automatically suggest a ranked list of mutations that are more likely to favour (or disfavour) particular couplings. Checkboxes are provided to enable the users to select the members of one or more G-protein families as target of the design. Residues of the input sequence corresponding to 7TM positions that are statistically associated to a coupling(s) of choice are systematically mutated into each of the remaining 19 amino acids and coupling probabilities computed. For each mutant and each G-protein coupling, two probability differences are calculated: P2RY8, a purinergic receptor which has been reported to be recurrently mutated in lymphomas, where it also displays mutual exclusivity with GNA13 . With the help of checkboxes, the user can also toggle between the significant positions of G-protein families to be displayed on the structure. Information about interaction contacts, either involving ligand or G-protein binding interfaces, or the network of intramolecular contacts, is obtained by our previous study  and can We compared the performance of PRECOG with that of PredCouple, a publicly available GPCR/G-protein prediction tool  by runniIndeed, our finally selected models outperformed both this last predictor as well as PredCouple, indicating the critical contribution from the TGFα shedding assay couplings .GNAI1/GNAI2 and GNAZ for CHRM3, and the predictor we have developed proved successful in reproducing them. PRECOG can also be used to illuminate the coupling mechanisms of poorly characterized receptors. For example, for the 61 receptors (21% of 286 Class A GPCRs) lacking coupling information from either GtoPdb or the chimeric G-protein-based assay, we predict a prevalence of Gs followed by Gq/11 and G12/13 couplings, the latter being the smallest fraction among currently known experimental couplings  – P(WT) ≥ 0.1). The most affected couplings are those of Gs, Gi/o and, to a lesser extent, G12/13 , and ma2/13 see .s complexes (i.e. PDB IDs: 3SN6 and 6GDG) are statistically associated to the corresponding couplings in the TGFα shedding assay (i.e. GNAS and GNAL), as well as the GNAO1-HTR1B (PDB ID: 6G79) complex is relevant for Gi/o couplings (i.e. GNAI1/GNAI2) (see i/o complexes (i.e. 6CMO and 6DDE) are also good templates to model G12/13 couplings, suggesting for this receptor class an interaction topology similar to the Gi/o family members  see . Surprisbers see .s family (q/11 or G12/13) will lead to additional improvements in the future.Integration of structure-derived features from 3D complex analysis leads to modest improvement of predictor performance only for the Gs family . It is lde novo for particular signalling effects.PRECOG represents a significant improvement over previous methods ,39 both GNA12 specific DREADD . Easy visualization of coupling determinants on sequence and structure, integrated with the usage of contact networks, that are increasingly employed to understand signalling protein mechanisms , will eac DREADD . Lastly,gkz392_Supplemental_FilesClick here for additional data file.


However, anticoagulation is not always achievable and up to 40% results in serious clotting in the venous expansion chamber. In this study, we have transferred one-quarter of the TSC from the prefiltered to the post filter based on RCA-one, which we have called RCA-two. The objective of this study was to compare the efficacy and safety of RCA-two with either saline flushing or RCA-one in HD patients with a high bleeding risk.Recommended regular saline flushing presents clinical ineffectiveness for hemodialysis (HD) patients at high risk of bleeding with heparin contraindication. Regional citrate anticoagulation (RCA) has previously been used with a CaIn this investigator-initiated, multicenter, controlled, prospective, randomized clinical trial, 52 HD patients (77 sessions) were randomized to the RCA-2 and RCA-one group in part one of the trial, and 45 patients (64 sessions) were randomized to the RCA-2 and saline group in part two of the trial. Serious clotting events, adverse events and blood analyses were recorded.Serious clotting events in the RCA-two group were significantly lower compared with the RCA-one and saline group . The median circuit survival time was 240 min (IQR 240 to 240) in the RCA-two group, was significantly longer than 230 min  in the RCA-one group and 210 min  in the saline group. The majority of the AEs were hypotension, hypoglycemia and chest tightness, most of which were mild in intensity. Eight patients (20.51%) in the RCA-one group, 4 patients (12.90%) in the saline group and 10 patients (26.31%) in the RCA-two group, P > 0.05.Our data demonstrated that the modified anticoagulation protocol was more effective and feasible during hemodialysis therapy for patients at high risk of bleeding.GDREC2017250H. Registered February 2, 2018; retrospectively registered.GDREC,  Chronic kidney disease (CKD) affects 10.8% of the population, and over 460,000 patients with CKD eventually suffer from chronic hemodialysis (HD) in China . Accurat2+) and magnesium (Mg2+) ions, the resulting absence of Ca2+ in the blood causes the anticoagulation effect. Usually an adequate amount of Ca2+ should be supplemented back to the systemic circulation according to the amount of imported TSC. Since 2012, RCA has been recommended as a priority anticoagulant for patients who require continuous renal replacement therapy (CRRT) because studies have shown that RCA has better anticoagulation properties and less bleeding risk than heparin 008), the Second Affiliated Hospital of Guangzhou Medicine University (Project Number: 2018-hs-06), and Guangzhou Hospital of Chinese Medicine (Project Number: B2017–189-01). The trial was prospectively registered at the international clinical trial registry system with the identifier GDREC2017250H .RCA-one group: A sterile solution of trisodium citrate  was continuously infused at the beginning of the arterial line. RCA-two group: 4% TSC was infused in two stages: at the beginning of the arterial line and at the venous expansion chamber . Generally, three-quarters of the total 4% TSC was infused at the beginning of the arterial line, and one-quarter was infused at the venous expansion chamber. The rate of 4% TSC was calculated according to the blood flow supplemented in Fig. 1c. Saline group: 0.9% saline flushes with 250 mL were carried out through the arterial line every 30 min .There were two parts to this trial. In part one, patients were randomized to the RCA-one or RCA-two group in a 1:1 ratio; in part two, patients were consecutively randomized to the Saline or RCA-two group in a 1:1 ratio Fig. .RCA-oneThe process of randomization was using concealed opaque envelopes. The randomization sequence was computer generated by a statistician. Sequence were enclosed in opaque sealed envelopes. After written informed consent was obtained, one envelope was randomly drawn and opened by the investigator. Patients were withdrawn from the study protocol when bleeding risk had disappeared or after completion of 3 sessions of dialysis treatment or discharged. Patients, treating physicians and nurses were not blinded to the patients’ treatment and Clotting of each circuit. Apart from the trial statistician and the data-monitoring committee, all treating physicians and nurses remained blinded to the trial results until this trial was completed.2, Nipro, Japan) or polyethersulfone (PES) membrane , which were reported to have similar thrombogenicity and sieving coefficients   in any position  in the RCA-two group was significantly lower than the RCA-one group and saline group . The majority of the AEs were hypotension and chest tightness, most of which were mild in intensity. In part two, seven patients (21.21%) in the RCA-two group and four patients (12.90%) in the saline group had complications (P = 0.379). A majority of the AEs were hypoglycemia and hypotension, most of which were mild to moderate in intensity.AEs are summarized in Table 2+) above 2.5. All T/I Ca2+ ratios at 0 h were less than 2.5. T/I Ca2+ ratios above 2.5 occurred in six of 71 patients (8.45%) at 4 h in the RCA-two group, two of 39 patients (5.13%) at 4 h in the RCA-one group . The change of ionized calcium serum levels from baseline to 4 h were not significant in two part respectively  was only found in a few patients throughout treatment, and there were no statistical differences in the occurrence of hypocalcemia among these groups  revealed fewer clotting events than RCA-one (RCA-Ca3), or anticoagulant-free hemodialysis, while Ca2+ supplementation is necessary for the RCA-Ca0 group [2+-containing dialysate or a hemodialysis filtration (HDF) procedure. Evenepoel et al. found that RCA-one for HD was shown to be safe and relatively effective but still exhibited moderate blood clotting compared with heparin  [2+, or blood platelets. Generally, RCA-one was inferior in the venous bubble trap, probably caused by an elimination of TSC by the dialyzer and restoration of Ca2+ by dialysate.Prior clinical studies have explored RCA under different conditions. An open-label single-center prospective clinical trial including 33 patients at risk of bleeding demonstrated that RCA using a Caa0 group . However= 0.036) . Later s= 0.036) . However2+ concentrations in the dialysate, wherein it was 1.5 mmol/L Ca2+ in our study, compared with 1.25 mmol/L Ca2+ in previous studies.To solve this problem, we modified the RCA-one technique, that one-quarter of the total TSC was imported from the artery line to the venous bubble trap. The strengths of this study included that it was a multicenter, randomized, prospective clinical trial. For the efficiency analysis, we compared RCA-two with saline flushing and RCA-one, and the anticoagulation superiority was significant in RCA-two group, comparing with the saline and RCA-one group. The anticoagulation effect in the RCA-one group in this study was worse than in previous studies , 25. OneDialysis session length is one of the essential factors associated with all-cause mortality among HD patients , 28, andAnother strength of this study was that major confounders were analyzed based on the Cox regression models, affirming the independent anticoagulation role of RCA-two Table . The pre2+ and rapidly producing bicarbonate [2+ concentration higher than 2.5 at 4 h. We also tested serum citrate concentration using a citrate assay kit, and the average serum citrate concentration of patients with a T/I Ca2+ concentration higher than 2.5 was 0.02 mmol/L (Range: 0.01–0.07 mmol/L), within the safety range (< 1.0 mmol/L) according to previous recommendations. The correlation of serum citrate and T/I Ca2+ was 0.537 (0.123–0.791), P = 0.015 (data not shown). Although several patients were at risk of citrate overdose by the end of therapy, the T/I Ca2+ recovery occurred in all of them by the beginning of the next HD session 2 or 3 days later. The exact recovery time is unknown, which is also a limitation of this study.Generally, statistical differences in the safety population is not expected given the size sample. So a larger clinical cohort was needed to provide more robust data about the safety population. One-quarter of the TSC was imported back to the systemic circulation in the RCA-two procedure. Is it safe enough, especially in patients with a high risk of bleeding? The TSC is mainly metabolized in the liver and skeletal muscle in HD patients, liberating the Caarbonate . Theoret2+, hypocalcemia is a major complication during RCA, which will subsequently cause hypotension and convulsions and even serious arrhythmia [2+ need not be monitored or supplemented into the systemic circulation. We determined whether the small portion of TSC imported directly back to the body in RCA-two would impact the serum Ca2+? As shown in Fig. 2+ was significantly lower in the RCA-two group, compared with the RCA-one and saline group, but the average concentration was above 0.9 mmol/L throughout the trial. Several patients developed asymptomatic hypocalcemia in RCA-two and RCA-one group demonstrated that patients were endurable with such complication. Hypomagnesemia, hypernatremia, and metabolic alkalosis were rare throughout the trial. Of note is that hyponatremia was observed in several participants, but we theorized that it was partly iatrogenic because we had reduced the concentration of sodium to 137 mmol/L in the dialysate. Other complications  did not differ among the groups. The major complication was hypotension, and this may be partly related to the larger ultrafiltration per hour with the infusion of TSC.Because TSC functions by binding Carhythmia . In the There are multiple limitations of this study that need to be mentioned. First, a larger clinical cohort is needed to provide more robust data about the safety population mentioned previously. Secondly, due to data limitations, we could not exclude all of the oral and hemostatic anticoagulants that participants were taking as a potential confounder and bias affecting the results , 33. OthP < 0.05; Additional file Third, there were several inevitable biases in this trial. All the hospitals participating in this trial were high-grade hospitals in the urban area. The basic homogeneity of the included participants was relatively stable, and the main safety assessment indexes may be better than those in the real world. In this study, participants could voluntarily take part in one to three sessions during the trial, and therefore, for the open label study design, a choice bias exists to some extent; for example, participants with serious clotting may not be willing to continue for another session. To minimize the bias, additional assessment in separated data including only the first session of all participants showed that serious clotting events in the RCA-two group were still significantly lower than in either the RCA-one or saline group.  or hemofiltration (HF) models, which are also major patterns of blood purification in our country.This study demonstrated the superiority of our modified anticoagulation RCA-two over either saline flushing or RCA-one in a population of intermittent HD patients with a high risk of hemorrhage. Our study clarified the effectiveness and safety of RCA-two. RCA-two could provide the best guarantee in dialysis sufficiency with the fewest clotting events and better clinical feasibility. At the same time, rare occurrences of TSC accumulation or other complications were found during the RCA-two procedure. Finally, a larger clinical cohort in the real world should be carried out in the future.Additional file 1: Figure S1. Efficacy endpoints in detail. The percentage of sessions with persistent venous pressure (> 200 mmHg), TMP (> 300 mmHg) and visible serious circuit clotting in part one (A) and part two (B); the clotting scores of ECC in part one (C) and part two (D). Abbreviations: Saline, saline flushing group; RCA-two, two-stage regional citrate anticoagulation group; RCA-one, one-stage regional citrate anticoagulation group; n.s., no significant statistical differences; TMP, transmembrane pressure.Additional file 2: Figure S2. Correlation coefficients between variables by Spearman’s correlation analysis in part one trial. The main purpose of this analysis was to reduce the possibility that the same associated variable entered the multivariate model simultaneously, the correlation analysis between the factors screened using univariate analysis was performed. If the Spearman’s correlation coefficient between variables exceeds 0.60, only the variables considering being more important on a clinical basis were entered into the multivariate model. Variables with similar functions were chosen using the likelihood ratio test, which favored those variables with higher statistical values. Abbreviations: APTT, Activated partial thromboplastin time.Additional file 3: Figure S3. Correlation coefficients between variables by Spearman’s correlation analysis in part two trial. The main purpose of this analysis was to reduce the possibility that the same associated variable entered the multivariate model simultaneously, the correlation analysis between the factors screened using univariate analysis was performed. If the Spearman’s correlation coefficient between variables exceeds 0.60, only the variables considering being more important on a clinical basis were entered into the multivariate model. Variables with similar functions were chosen using the likelihood ratio test, which favored those variables with higher statistical values. Abbreviations: APTT, Activated partial thromboplastin time.Additional file 4: Figure S4. Efficacy endpoints of the first session of all patients. (A) Serious clotting rate in part one and part two trial (9/26(34.62%) vs. 2/26(7.69%), P = 0.039; 11/22(50.00%) vs. 1/23(4.35%), P < 0.001, respectively). (B) The circuit survival time of part one and part two. The median circuit survival time was 240 min (IQR 173 to 240) in the RCA-one group vs 240 min (IQR 240 to 240) in the RCA-two group (P = 0.019); The median circuit survival time was 221 min (IQR 170 to 240) in the Saline group vs 240 min (IQR 240 to 240) in the RCA-two group (P < 0.001). Abbreviations: Saline, saline flushing group; RCA-two, two-stage regional citrate anticoagulation group; RCA-one, one-stage regional citrate anticoagulation group.Additional file 5: Figure S5. The concentration of iCa2+ at several points of the ECC in RCA-tw o (A) and RCA-one (B). (A) We innovatively transferred one-quarter of the TSC from the prefilter to the postfilter based on RCA-one, which is called RCA-two in this trial. The concentration of iCa2+ at venous bubble trap was reduced significantly. (B) We previously tested the concentration of iCa2+ at several points of the ECC in RCA-one and found that the concentration of iCa2+ is particularly high in the venous bubble trap, which would be the key point of clotting. Abbreviations: RCA, regional citrate anticoagulation; ECC, the extracorporeal circuit; TSC, trisodium citrate.Additional file 6: Table S1. Criteria of clotting scores of extra corporeal circuit at the end of dialysis.Additional file 7: Table S2. Supplemental baseline characteristics.Additional file 8: Table S3. Univariate analysis of influencing factors of serious clotting events.